JP6731587B2 - Photosensitive composition, patterned substrate, cell culture support and method for producing cultured cells - Google Patents

Description

The present invention relates to a photosensitive composition, a patterned substrate, a cell culture support and a method for producing cultured cells.

Poly(N-isopropylacrylamide) and the like are known as temperature-responsive polymers that dissolve in water below the lower critical point temperature (LCST) and do not dissolve in water at higher temperatures. A technique for producing a cell culture support using such a temperature-responsive polymer is known (for example, Non-Patent Document 1). In a cell culture support produced using a temperature-responsive polymer, cells attached to the support can be exfoliated by adjusting the temperature.

A technique for producing a chip having a dot pattern formed by a photolithography method using a photosensitive composition containing a temperature responsive polymer is known. For example, in Patent Document 1, the following formula:

(In the above formula, R ₁ and R ₂ represent a hydrogen atom or a C _1-4 alkyl group, and R ₃ represents a hydrocarbon structure capable of being crosslinked with the crosslinking agent.)
There is described a photosensitive composition containing an N-alkylacrylamide copolymer having a repeating unit represented by, a crosslinking agent and an acid generator. As represented by the above formula, the N-alkylacrylamide copolymer is a binary copolymer composed of a structural unit derived from N-alkylacrylamide and a crosslinkable structural unit (R ₃ portion).

International Publication No. 2005/095510

Y. Tsuda et al., Biochemical and Biophysical Research Communications 348 (2006) 937-944

Various temperature-responsive patterned substrates (especially cell culture supports) having different physical properties, for example, the temperature at which the hydrophilicity and hydrophobicity of the pattern change (hereinafter abbreviated as “response temperature”) are subjected to temperature response. It is required to manufacture a photosensitive composition containing a photosensitive polymer by patterning by a photolithography method. This response temperature can be changed by changing the composition of the temperature-responsive polymer used and adjusting its lower critical point temperature (LCST). However, in the binary copolymer as described in Patent Document 1, when the composition is changed to adjust the LCST, the amount of the crosslinkable constitutional unit is insufficient, and patterning by the photolithography method cannot be performed. There are cases.

The present invention has been made in view of the above circumstances, and an object thereof is to produce a temperature-responsive patterned substrate (in particular, a cell culture support) having a desired response temperature by a photolithography method. To provide a photosensitive composition capable of

As a result of intensive studies by the present inventors, a temperature-responsive polymer containing a structural unit represented by the following formulas (1) to (3) (that is, a copolymer containing at least three types of structural units) is used. For example, the temperature can be adjusted to a desired lower critical point temperature (LCST) without impairing the crosslinkability (patterning property), and the temperature having a desired response temperature can be obtained from the photosensitive composition containing the temperature responsive polymer. It has been found that responsive patterned substrates (particularly cell culture supports) can be produced by patterning by photolithography. The present invention based on this finding is as follows.

[1] Formula (1):

(In the formula, R ¹¹ represents a hydrogen atom or a methyl group.
R ¹² and R ¹³ each independently represent a C _1-4 alkyl group.
* Indicates a binding position. )
A structural unit represented by (hereinafter sometimes abbreviated as “structural unit (1)”),
Formula (2):

(In the formula, R ²¹ represents a hydrogen atom or a methyl group.
R ²² represents a ^{-CO-O-R 23} or ^-CO-NR 24 ^{R 25.}
R ²³ represents a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3 to 10-membered heterocyclic group having a reactive group.
One of R ²⁴ and R ²⁵ is a hydrogen atom, an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3 to 10 Membered heterocyclic group, the remaining one is a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3-10 membered heterocyclic group having a reactive group. Indicates.
* Indicates a binding position. )
A structural unit represented by (hereinafter sometimes abbreviated as “structural unit (2)”), and a formula (3):

(In the formula, R ³¹ represents a hydrogen atom or a methyl group.
R ³² represents —CO—O—R ³³ or —CO—NR ³⁴ R ³⁵ .
R ³³ represents an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3-10 membered heterocyclic group.
R ³⁴ and R ³⁵ are each independently a hydrogen atom, an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3 to 3 group. A 10-membered heterocyclic group is shown.
* Indicates a binding position. )
(Except those which are the same as the constitutional unit represented by formula (1) and the constitutional unit represented by formula (2)) (hereinafter, abbreviated as “constitutional unit (3)”) Sometimes, a copolymer containing
A photosensitive composition containing a photoacid generator and a solvent.
[2] The photosensitive composition according to the above [1], wherein R ²² is —CO—O—R ²³ , and R ²³ is a C _1-4 alkyl group having a reactive group.
[3] The photosensitive composition according to the above [1] or [2], wherein the reactive group is a hydroxy group.
^{[4] R 33} is optionally having hydroxy group _{C 2-10} alkyl group or ^-R 36 ^-R 37 ^{-R 38} (in the ^{formula, R 36} _is a _{C 1-4} alkylene group, R ³⁷ is a C _3-6 cycloalkanediyl group, R ³⁸ is a C _1-4 alkyl group _optionally having a hydroxy group), and one of R ³⁴ and R ³⁵ is The photosensitive composition according to any one of the above [1] to [3], which is a C _3-10 alkyl group, and the other one is a hydrogen atom or a C _1-10 alkyl group.
[5] The constitutional unit represented by the formula (3) includes ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, sec-butyl (meth). ) Acrylate, tert-butyl (meth)acrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 1,4-cyclohexanedimethanol mono(meth)acrylate, N- Derived from a monomer selected from the group consisting of propyl(meth)acrylamide, N-butyl(meth)acrylamide, N-(tert-butyl)(meth)acrylamide, and N-methyl-N-isopropyl(meth)acrylamide. The photosensitive composition according to any one of the above [1] to [3], which is a structural unit.
[6] The photosensitive composition according to the above [4] or [5], wherein the lower critical point temperature of the copolymer is 15°C or higher and lower than 33°C.
[7] R ³² is —CO—NR ³⁴ R ³⁵ , and R ³⁴ and R ³⁵ are each independently a C _1-3 alkyl group having a hydrogen atom, a methyl group or an ethyl group, or a hydroxy group. The photosensitive composition as described in any one of [1] to [3] above.
[8] The structural unit represented by the formula (3) is acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-ethyl-N-methylacrylamide, Any of the above-mentioned [1] to [3], which is a structural unit derived from a monomer selected from the group consisting of N-(2-hydroxyethyl)acrylamide and N-(2-hydroxy-1-methylethyl)acrylamide. The photosensitive composition as described in 1 above.
[9] The photosensitive composition according to the above [7] or [8], wherein the lower critical point temperature of the copolymer is 33 to 45°C.
[10] In all the constituent units of the copolymer, the amount of the constituent unit represented by the formula (1) is 10 to 94 mol %, and the amount of the constituent unit represented by the formula (2) is 5 to 40 mol. %, and the amount of the structural unit represented by the formula (3) is 1 to 80 mol %, wherein the photosensitive composition according to any one of [1] to [9].
[11] The photosensitive composition according to any one of the above [1] to [10], further containing a crosslinking agent that reacts with a reactive group in the presence of an acid.
[12] The photosensitive composition according to any one of [1] to [11], which is used for producing a cell culture support.
[13] A patterned substrate formed from the photosensitive composition according to any one of [1] to [11].
[14] A coating film is formed on a substrate by using the photosensitive composition according to any one of [1] to [11], and the coating film is irradiated with light to form a coating film after exposure. A method of manufacturing a patterned substrate, comprising a step of developing.
[15] A coating film is formed on a substrate by using the photosensitive composition according to any one of [1] to [11], and the coating film is irradiated with light to form a coating film after exposure. A pattern forming method including a step of developing.

[16] A photosensitive composition containing the temperature-responsive polymer (I) is used to form a coating film on a substrate, the coating film is irradiated with light, and the exposed coating film is developed to form a pattern. Forming a coating film on a substrate by using the photosensitive composition containing the temperature-responsive polymer (II) having a lower critical point temperature different from that of the temperature-responsive polymer (I) A step of irradiating the coating film with light and developing the coating film after exposure to form a pattern (II)
A method for producing a cell culture support, comprising:
[17] The production method according to the above [16], wherein the photosensitive composition further contains a photoacid generator and a solvent.
[18] The production method according to the above [16] or [17], wherein the photosensitive composition further contains a crosslinking agent.
[19] One of the temperature-responsive polymer (I) and the temperature-responsive polymer (II) is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3), and the temperature response is The other of the water-soluble polymer (I) and the temperature-responsive polymer (II) is a copolymer containing the structural unit (1) and the structural unit (2) but not the structural unit (3), or [16], in which the responsive polymer (I) and the temperature responsive polymer (II) are each independently a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3). ~ The manufacturing method according to any one of [18].
[20] The production method according to any one of the above [16] to [19], wherein in the step (I), post exposure bake is performed at a temperature of 40 to 200°C.
[21] The manufacturing method according to any one of [16] to [20], in which the pattern formed in step (I) is irradiated with light.
[22] The manufacturing method according to any one of [16] to [21], wherein the formed pattern has a thickness of 1 to 100 nm.
[23] A cell culture support obtained by the production method according to any one of [16] to [22].

[24] A substrate and a cell culture support having a plurality of patterns on the substrate, wherein at least one pattern is formed from a photosensitive composition containing a temperature responsive polymer (I), A cell culture support, wherein at least one other pattern is formed from a photosensitive composition containing a temperature-responsive polymer (II) having a lower critical point temperature different from that of the temperature-responsive polymer (I).
[25] The cell culture support according to the above [24], wherein the photosensitive composition further contains a photoacid generator and a solvent.
[26] The cell culture support according to the above [24] or [25], wherein the photosensitive composition further contains a crosslinking agent.
[27] One of the temperature-responsive polymer (I) and the temperature-responsive polymer (II) is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3), and the temperature response is The other of the water-soluble polymer (I) and the temperature-responsive polymer (II) is a copolymer containing the structural unit (1) and the structural unit (2) but not the structural unit (3), or the temperature [24], in which the responsive polymer (I) and the temperature responsive polymer (II) are each independently a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3). ~ The cell culture support according to any one of [26].
[28] The cell culture support according to any one of [24] to [27], wherein the formed pattern has a thickness of 1 to 100 nm.

[29] A substrate and a cell culture support having a plurality of patterns on the substrate,
At least one pattern includes a crosslinked product of the temperature-responsive polymer (I), and at least one other pattern is formed of a temperature-responsive polymer (II) having a lower critical point temperature different from that of the temperature-responsive polymer (I). Including crosslinked products,
One of the temperature responsive polymer (I) and the temperature responsive polymer (II) is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3), and the temperature responsive polymer ( The other of I) and the temperature responsive polymer (II) is a copolymer containing the structural unit (1) and the structural unit (2) and not the structural unit (3), or the temperature responsive polymer A cell culture support, wherein (I) and temperature-responsive polymer (II) are copolymers each independently containing structural unit (1), structural unit (2), and structural unit (3).
[30] The cell culture support according to the above [29], wherein the pattern has a thickness of 1 to 100 nm.

[31] A method for producing cultured cells using a substrate and a cell culture support having a plurality of patterns on the substrate, the method comprising:
At least one pattern (I) is formed from a photosensitive composition containing a temperature responsive polymer (I), and at least one other pattern (II) is a temperature responsive polymer (I). It is formed from a photosensitive composition containing a temperature responsive polymer (II) having a lower critical point temperature higher than the lower critical point temperature thereof.
A cell (on at least one pattern (I)) at a temperature higher than the lower critical point temperature of the temperature responsive polymer (I) and lower than the lower critical point temperature of the temperature responsive polymer (II). And culturing the cells (I) at a temperature higher than the lower critical point temperature of the temperature-responsive polymer (I) and the temperature-responsive polymer (II) on at least one other pattern (II). The manufacturing method including the process of culturing the cell (II) different from the above.
[32] Furthermore, the temperature of the pattern (I) and the pattern (II) is adjusted to a temperature lower than the lower critical point temperature of the temperature-responsive polymer (I) and the temperature-responsive polymer (II), and cell culture is performed. The production method according to the above [31], which comprises a step of separating cells (I) and cells (II) from the support.
[33] The production method according to the above [31] or [32], wherein the photosensitive composition further contains a photoacid generator and a solvent.
[34] The production method according to any one of [31] to [33], wherein the photosensitive composition further contains a crosslinking agent.
[35] One of the temperature-responsive polymer (I) and the temperature-responsive polymer (II) is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3), and the temperature response is The other of the water-soluble polymer (I) and the temperature-responsive polymer (II) is a copolymer containing the structural unit (1) and the structural unit (2) but not the structural unit (3), or the temperature [31], wherein the responsive polymer (I) and the temperature responsive polymer (II) are each independently a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3). ~ The production method according to any one of [34].

[36] A method for producing cultured cells using a substrate and a cell culture support having a plurality of patterns on the substrate, the method comprising:
At least one pattern (I) includes a crosslinked product of the temperature responsive polymer (I), and at least one other pattern (II) has a lower limit higher than the lower critical point temperature of the temperature responsive polymer (I). A crosslinked product of a temperature responsive polymer (II) having a critical point temperature,
One of the temperature responsive polymer (I) and the temperature responsive polymer (II) is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3), and the temperature responsive polymer ( The other of I) and the temperature responsive polymer (II) is a copolymer containing the structural unit (1) and the structural unit (2) and not the structural unit (3), or the temperature responsive polymer (I) and the temperature-responsive polymer (II) are copolymers each independently containing the structural unit (1), the structural unit (2), and the structural unit (3),
A cell (on at least one pattern (I)) at a temperature higher than the lower critical point temperature of the temperature responsive polymer (I) and lower than the lower critical point temperature of the temperature responsive polymer (II). And culturing the cells (I) at a temperature higher than the lower critical point temperature of the temperature-responsive polymer (I) and the temperature-responsive polymer (II) on at least one other pattern (II). The manufacturing method including the process of culturing the cell (II) different from the above.
[37] Furthermore, the temperature of the pattern (I) and the pattern (II) is adjusted to a temperature lower than the lower critical point temperature of the temperature-responsive polymer (I) and the temperature-responsive polymer (II), and cell culture is performed. [36] The production method according to the above [36], which comprises a step of separating cells (I) and cells (II) from the support.

[38] A cured product obtained from the photosensitive composition according to any one of [1] to [12].

[39] A cell culture support comprising the steps of forming a coating film on a substrate using a photosensitive composition containing a temperature-responsive polymer and irradiating the coating film with light to form a cured product layer. A manufacturing method,
The production method, wherein the temperature-responsive polymer is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3).

[40] A cell culture support having a substrate and a cured product layer formed from a photosensitive composition containing a temperature-responsive polymer on the substrate,
The cell culture support, wherein the temperature-responsive polymer is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3).

[41] A cell culture support having a substrate and a cured product layer containing a crosslinked product of a temperature-responsive polymer on the substrate,
The cell culture support, wherein the temperature-responsive polymer is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3).

[42] A method for producing a cultured cell using a substrate and a cell culture support having a cured product layer on the substrate, the method comprising:
The cured product layer is formed from a photosensitive composition containing a temperature responsive polymer,
The temperature responsive polymer is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3),
A production method comprising a step of culturing cells on a cured product layer at a temperature higher than a lower critical point temperature of a temperature-responsive polymer.

[43] A method for producing cultured cells using a substrate and a cell culture support having a cured product layer on the substrate, the method comprising:
The cured product layer contains a crosslinked product of a temperature responsive polymer,
The temperature responsive polymer is a copolymer containing the structural unit (1), the structural unit (2), and the structural unit (3),
A production method comprising culturing cells on a cured product layer at a temperature higher than a lower critical point temperature of a temperature-responsive polymer.

By using the photosensitive composition of the present invention, a temperature-responsive patterned substrate (in particular, a cell culture support) having a desired response temperature can be produced by patterning by a photolithography method.

3 is a graph showing the results of a sensitivity test of the photosensitive composition obtained in Example 1. 5 is a graph showing the results of a sensitivity test of the photosensitive composition obtained in Example 2. 5 is a graph showing the results of a sensitivity test of the photosensitive composition obtained in Example 3. 5 is a graph showing the results of a sensitivity test of the photosensitive composition obtained in Example 4. 5 is a graph showing the results of a sensitivity test of the photosensitive composition obtained in Example 5. 5 is a graph showing the results of a sensitivity test of the photosensitive composition obtained in Comparative Example 1. 3 is a graph showing the results of a patterning test of the photosensitive composition obtained in Example 1. 5 is a graph showing the results of a patterning test of the photosensitive composition obtained in Example 2. 5 is a graph showing the results of a patterning test of the photosensitive composition obtained in Example 3. 5 is a graph showing the results of a patterning test of the photosensitive composition obtained in Example 4. 5 is a graph showing the results of a patterning test of the photosensitive composition obtained in Comparative Example 1. 9 is a schematic plan view of patterns Ia, Ib, IIa, and IIb in the cell culture support 2 obtained in Example 7. FIG.

<Photosensitive composition and cured product obtained therefrom>
The photosensitive composition of the present invention contains a copolymer containing the structural units (1) to (3), a photoacid generator, and a solvent. The photosensitive composition of the present invention preferably further contains a crosslinking agent that reacts with the reactive group in the presence of an acid. In the copolymer, each of the structural units (1) to (3) may be only one type, or may be two or more types. Hereinafter, the groups contained in the structural units (1) to (3) will be described in order.

In the present specification, “C _ab ” (in the above formula, a and b represent integers) means that the carbon number is ab.

In the present specification, the “reactive group” means a hydroxy group, a sulfanyl group, a carboxy group or a C _1-4 alkoxymethyl group. From the viewpoint of reactivity, the reactive group is preferably a hydroxy group.

In the present specification, the alkyl group may be linear or branched. Examples of the "C _1-10 alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl. , Decyl.

In the present specification, the “C _1-3 alkyl group”, “C _1-4 alkyl group”, “C _2-10 alkyl group” and “C _3-10 alkyl group” are the above-mentioned “C _1-10 ”. In the examples of the "alkyl group", those having 1 to 3 carbon atoms, those having 1 to 4 carbon atoms, those having 2 to 10 carbon atoms, and those having 3 to 10 carbon atoms, respectively. The thing is illustrated.

In the present specification, examples of the “C _3-10 cycloalkyl group” include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl.

In the present specification, the “3- to 10-membered heterocyclic group” includes, for example, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and the like. A group; and aziridinyl, oxiranyl, thiranyl, azetidinyl, oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, hydrozolinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiazolinyl, , Tetrahydroisoxazolyl, piperidinyl, piperazinyl, tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, Examples thereof include non-aromatic heterocyclic groups such as thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl, azocanyl and diazocanyl.

In the present specification, the alkylene group may be linear or branched. Examples of the “C _1-4 alkylene group” include —CH ₂ —, —(CH ₂ ) ₂ —, —(CH ₂ ) ₃ —, —(CH ₂ ) ₄ —, —CH(CH ₃ )—, _{-C (CH 3) 2 -,} - CH (C 2 H 5) -, - CH (C 3 H 7) -, - CH (CH (CH 3) 2) -, - (CH (CH 3)) 2 _{-, - CH 2 -CH (CH} 3) -, - CH (CH 3) -CH 2 - and the like.

In the present specification, examples of the “C _3-6 cycloalkanediyl group” include cyclopropane-1,2-diyl, cyclobutane-1,3-diyl, cyclopentane-1,3-diyl, cyclohexane-1, 4-diyl is mentioned.

In the present specification, examples of the substituent that the “optionally substituted alkyl group” and the “optionally substituted cycloalkyl group” may have include, for example, a halogen atom, an amino group, a mono- or di-C. Examples thereof include a _1-4 alkylamino group, a nitro group, a cyano group, an oxo group, a hydroxy group, a sulfanyl group, a C _1-4 alkoxy group, a C _1-4 alkoxycarbonyl group, a formyl group and a C _6-14 aryl group.

In the present specification, examples of the substituent that the “optionally substituted heterocyclic group” may have include a halogen atom, an amino group, a mono- or di-C _1-4 alkylamino group, a nitro group, and a cyano group. Examples thereof include a group, an oxo group, a hydroxy group, a sulfanyl group, a C _1-4 alkoxy group, a C _1-4 alkoxycarbonyl group, a formyl group and a C _1-10 alkyl group.

In the present specification, examples of the “halogen atom” include fluorine, chlorine, bromine and iodine.

In the present specification, examples of the “mono- or di-C _1-4 alkylamino group” include methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino. , N-ethyl-N-methylamino.

In the present specification, the alkoxy group may be linear or branched. Examples of the "C _1-4 alkoxy group" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy.

In the present specification, examples of the “C _6-14 aryl group” include phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl and 9-anthryl.

R ¹¹ in the structural unit (1) is a hydrogen atom or a methyl group, preferably a hydrogen atom.
R ¹² and R ¹³ are each independently a C _1-4 alkyl group, preferably a methyl group.

The structural unit (1) is preferably a structural unit derived from N-isopropylacrylamide. Here, the constitutional unit derived from N-isopropylacrylamide means a constitutional unit formed by polymerization of carbon-carbon double bonds of N-isopropylacrylamide, and may be one directly formed from N-isopropylacrylamide. Alternatively, it may be one indirectly formed from another monomer (for example, one obtained by forming a constitutional unit by polymerization of an N-isopropylacrylamide derivative and then converting the constitutional unit). The meaning of "derived from" is the same for the structural units (2) and (3).

R ²¹ in the structural unit (2) is a hydrogen atom or a methyl group, preferably a hydrogen atom.
R ²² is ^{-CO-O-R 23} or ^-CO-NR 24 ^{R 25.}
R ²³ is a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3-10 membered heterocyclic group having a reactive group.
One of R ²⁴ and R ²⁵ is a hydrogen atom, an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3 to 10 Membered heterocyclic group, the other one is a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3-10 membered heterocyclic group having a reactive group. Is.

The structural unit (2) is a crosslinkable structural unit having a reactive group on the side chain R ²² . When two or more kinds of structural units (2) are contained in the copolymer and they have a reactive group which reacts with each other, a cross-linking structure can be formed by their reaction. When the structural unit (3) has a substituent that reacts with the reactive group, a crosslinked structure can be formed by the reaction of the reactive group and the substituent. Further, when the photosensitive composition of the present invention contains a crosslinking agent, a crosslinked structure can be formed by the reaction between the reactive group and the crosslinking agent.

R ²² is preferably —CO—O—R ²³ (In the above formula, R ²³ is preferably a C _1-4 alkyl group having a reactive group, and more preferably C _1-4 having a hydroxy group. An alkyl group, more preferably 1-hydroxymethyl or 2-hydroxyethyl, most preferably 2-hydroxyethyl), or -CO-NR ²⁴ R ²⁵ (wherein R ²⁴ is preferably Is a hydrogen atom, R ²⁵ is preferably a C _1-10 alkyl group having a reactive group, more preferably a C _1-4 alkyl group having a reactive group, and further preferably a hydroxy group. It is a C _1-4 alkyl group, most preferably 2-hydroxy-1-methylethyl).

R ²² is more preferably —CO—O—R ²³ (in the above formula, R ²³ is preferably a C _1-4 alkyl group having a reactive group, and more preferably C _1-4 having a hydroxy group. It is an alkyl group, more preferably 1-hydroxymethyl or 2-hydroxyethyl, most preferably 2-hydroxyethyl.).

The structural unit (2) is preferably a structural unit derived from a monomer selected from the group consisting of 2-hydroxyethyl acrylate and N-(2-hydroxy-1-methylethyl)acrylamide, and more preferably 2 -A constitutional unit derived from hydroxyethyl acrylate.

R ³¹ in the structural unit (3) is a hydrogen atom or a methyl group.
R ³² is —CO—O—R ³³ or —CO—NR ³⁴ R ³⁵ .
R ³³ is an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3-10 membered heterocyclic group.
R ³⁴ and R ³⁵ are each independently a hydrogen atom, an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3 to 3 group. It is a 10-membered heterocyclic group.
However, in the present invention, the structural unit (3) is different from the above structural units (1) and (2).

The structural unit (3) different from the structural units (1) and (2) is mainly used for adjusting the lower critical point temperature (LCST) of the copolymer. In addition to the structural units (1) and (2), the LCST of the copolymer containing the structural unit (3), which is more hydrophobic than the structural units (1) and (2), is a binary copolymer of the structural units (1) and (2). It tends to be lower than the combined LCST. On the contrary, the LCST of the copolymer containing the structural unit (3), which is more hydrophilic in addition to the structural units (1) and (2), consists of the structural units (1) and (2). It tends to be higher than the LCST of the binary copolymer.

The value of the lower critical point temperature (LCST) of the copolymer in the present invention is one of two containers of a differential scanning calorimetry (DSC) aqueous solution of the copolymer (copolymer concentration: 2% by weight, solvent: pure water), the temperature of the solution was lowered at 1° C./min, and the temperature at the peak top of the measurement curve obtained by measuring the differential heat with an empty container containing only air as a control Is calculated as

In the present invention, a copolymer having a lower critical point temperature (LCST) of 15° C. or more and less than 33° C. (hereinafter abbreviated as “copolymer for low temperature”) and a copolymer having an LCST of 33 to 45° C. (Hereinafter, abbreviated as "copolymer for high temperature") can be used. The LCST of the low temperature copolymer is preferably 20° C. or higher and lower than 33° C., and the high temperature copolymer LCST is preferably 33 to 40° C.

The lower criticality of the copolymer measured in the same manner as described above, except that an aqueous solution of the copolymer, which is a phosphate buffered saline solution, is used instead of pure water (copolymer concentration: 2% by weight). The value of the point temperature (LCST) is preferably 10° C. or higher and lower than 29° C., more preferably 20° C. or higher and lower than 29° C. for low temperature copolymers, and preferably 29 to 29° C. for high temperature copolymers. 42 degreeC, More preferably, it is 29-40 degreeC. The lower critical point temperature (LCST) of the copolymer in the present invention is a value measured using pure water as a solvent, unless otherwise specified.

Hereinafter, the preferable structural unit (3) in the low temperature copolymer will be described in order.
In the copolymer for low temperature, R ³³ in the structural unit (3) is a C _2-10 alkyl group which may have a hydroxy group or —R ³⁶ —R ³⁷ —R ³⁸ (wherein R 3 is the above formula). ³⁶ is a C _1-4 alkylene group, R ³⁷ is a C _3-6 cycloalkanediyl group, and R ³⁸ is a C _1-4 alkyl group which may have a hydroxy group.) And one of R ³⁴ and R ³⁵ is a C _3-10 alkyl group, and the remaining one is preferably a hydrogen atom or a C _1-10 alkyl group.

In the low temperature copolymer, the structural unit (3) is preferably ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, sec-. Butyl(meth)acrylate, tert-butyl(meth)acrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropyl(meth)acrylate, 4-hydroxybutyl(meth)acrylate, 1,4-cyclohexanedimethanol mono(meth)acrylate , A monomer selected from the group consisting of N-propyl(meth)acrylamide, N-butyl(meth)acrylamide, N-(tert-butyl)(meth)acrylamide, and N-methyl-N-isopropyl(meth)acrylamide. Is a structural unit derived from. In addition, in this specification, "(meth)acrylate" etc. mean "acrylate and methacrylate" etc.

In the copolymer for low temperature, the constituent unit (3) is more preferably 2-hydroxyethyl methacrylate, 3-hydroxypropyl acrylate, 4-hydroxybutyl acrylate, 1,4-cyclohexanedimethanol monoacrylate, N-propyl. It is a structural unit derived from a monomer selected from the group consisting of acrylamide, N-butylacrylamide, N-(tert-butyl)acrylamide, and N-methyl-N-isopropylacrylamide.

In the low temperature copolymer, the structural unit (3) is more preferably a structural unit derived from N-propylacrylamide.

Next, the combination of structural units in the low temperature copolymer will be described.
In the low temperature copolymer, the structural unit (1) is a structural unit derived from N-isopropylacrylamide, and the structural unit (2) is 2-hydroxyethyl acrylate and N-(2-hydroxy-1-methylethyl). ) A structural unit derived from a monomer selected from the group consisting of acrylamide, and the structural unit (3) includes ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, Isobutyl (meth)acrylate, sec-butyl (meth)acrylate, tert-butyl (meth)acrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 1,4- From cyclohexanedimethanol mono(meth)acrylate, N-propyl(meth)acrylamide, N-butyl(meth)acrylamide, N-(tert-butyl)(meth)acrylamide, and N-methyl-N-isopropyl(meth)acrylamide It is preferably a constitutional unit derived from a monomer selected from the group consisting of:

In the low temperature copolymer, the structural unit (1) is a structural unit derived from N-isopropylacrylamide, and the structural unit (2) is 2-hydroxyethyl acrylate and N-(2-hydroxy-1-methylethyl). ) A structural unit selected from the group consisting of acrylamide, and the structural unit (3) is 2-hydroxyethyl methacrylate, 3-hydroxypropyl acrylate, 4-hydroxybutyl acrylate, 1,4-cyclohexanedimethanol monoacrylate, N-propyl. It is more preferable that the structural unit is derived from a monomer selected from the group consisting of acrylamide, N-butylacrylamide, N-(tert-butyl)acrylamide, and N-methyl-N-isopropylacrylamide.

In the low temperature copolymer, the structural unit (1) is a structural unit derived from N-isopropylacrylamide, and the structural unit (2) is 2-hydroxyethyl acrylate and N-(2-hydroxy-1-methylethyl). ) It is a structural unit selected from the group consisting of acrylamide, and it is more preferable that the structural unit (3) is a structural unit derived from N-propylacrylamide.

Hereinafter, the preferable structural unit (3) in the high temperature copolymer will be described in order.
In the copolymer for high temperature, R ³² in the structural unit (3) is —CO—NR ³⁴ R ³⁵ , and R ³⁴ and R ³⁵ are each independently a hydrogen atom, a methyl group or an ethyl group, or It is preferably a C _1-3 alkyl group having a hydroxy group.

In the high temperature copolymer, the structural unit (3) is preferably acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-ethyl-N-. It is a structural unit derived from a monomer selected from the group consisting of methylacrylamide, N-(2-hydroxyethyl)acrylamide, and N-(2-hydroxy-1-methylethyl)acrylamide.

In the copolymer for high temperature, the structural unit (3) is more preferably a unit amount selected from the group consisting of acrylamide, N,N-dimethylacrylamide, and N-(2-hydroxy-1-methylethyl)acrylamide. It is a structural unit derived from the body.

Next, the combination of structural units in the high temperature copolymer will be described.
In the high temperature copolymer, the structural unit (1) is a structural unit derived from N-isopropylacrylamide, and the structural unit (2) is 2-hydroxyethyl acrylate and N-(2-hydroxy-1-methylethyl). ) A structural unit derived from a monomer selected from the group consisting of acrylamide, the structural unit (3) being acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, N,N-diethyl. A structural unit derived from a monomer selected from the group consisting of acrylamide, N-ethyl-N-methylacrylamide, N-(2-hydroxyethyl)acrylamide, and N-(2-hydroxy-1-methylethyl)acrylamide. It is preferable to have.

In the high temperature copolymer, the structural unit (1) is a structural unit derived from N-isopropylacrylamide, the structural unit (2) is a structural unit derived from 2-hydroxyethyl acrylate, and the structural unit (3 ) Is more preferably a structural unit derived from a monomer selected from the group consisting of acrylamide, N,N-dimethylacrylamide, and N-(2-hydroxy-1-methylethyl)acrylamide.

The amount of the structural unit (1) in all the structural units of the copolymer is preferably 10 to 94 mol %, more preferably 20 to 90 mol% for adjusting the LCST and for increasing the phase transition enthalpy change. , And more preferably 40 to 80 mol %.
The amount (2) of the structural units in all the structural units of the copolymer is preferably 5 to 40 mol %, more preferably 7 to 30 mol %, and further preferably 10 to 20 in order to achieve sufficient crosslinking. Mol%.
The amount of the structural unit (3) in all the structural units of the copolymer is preferably 1 to 80 mol%, more preferably 2 to 60 mol%, and further preferably 3 to 40 mol% for LCST adjustment. is there.

The copolymer may contain a structural unit other than the structural units (1) to (3) as long as the temperature response and the crosslinkability (patterning property) are not impaired. The amount of the structural units other than the structural units (1) to (3) in all the structural units of the copolymer is preferably 20 mol% or less, more preferably 10 mol% or less. The copolymer is more preferably composed of structural units (1) to (3).

The weight average molecular weight (Mw) of the copolymer is preferably 500 to 1,000,000, more preferably 1,000 to 500,000, further preferably 3,000 to 100 in order to obtain good patterning characteristics. 1,000. The Mw value of the copolymer in the present invention is measured by gel permeation chromatography (GPC).

The content of the copolymer is preferably 50 to 99% by weight, more preferably 50 to 99% by weight, based on the solid content of the photosensitive composition excluding the solvent, in order to achieve good patterning characteristics and good expression of LCST. It is preferably 60 to 97% by weight, more preferably 80 to 95% by weight.

The above-mentioned copolymer can be produced by polymerizing a commercially available monomer or a monomer produced by a known method by a known method.

The photosensitive composition of the present invention contains a photoacid generator. The photoacid generator may be used alone or in combination of two or more.

The photo-acid generator is not particularly limited, and those commonly used in the field of photosensitive compositions can be used in the present invention. Examples of the photoacid generator include diazomethane compounds, onium salt compounds, sulfonimide compounds, nitrobenzyl compounds, iron arene complexes, benzointosylate compounds, halogen-containing triazine compounds, cyano group-containing oxime sulfonate compounds, and naphthalimide compounds. Are listed.

Examples of the diazomethane compound include bis(p-toluenesulfonyl)diazomethane, bis(1,1-dimethylethylsulfonyl)diazomethane, bis(cyclohexylsulfonyl)diazomethane, and bis(2,4-dimethylphenylsulfonyl)diazomethane.

Examples of the onium salt compound include bis(4-tert-butylphenyl)iodonium trifluoromethanesulfonate and triphenylsulfonium trifluoromethanesulfonate.

Examples of the sulfonimide compound include N-(trifluoromethanesulfonyloxy)succinimide, N-(nonafluoro-normal butanesulfonyloxy)succinimide, N-(camphorsulfonyloxy)succinimide, and N-(trifluoromethanesulfonyloxy)naphthalimide. Can be mentioned.

Examples of the nitrobenzyl compound include 2-nitrobenzyl p-toluenesulfonate, 2,6-dinitrobenzyl p-toluenesulfonate, and 2,4-dinitrobenzyl p-toluenesulfonate.

Examples of the iron arene complex include biscyclopentadienyl-(η ⁶ -isopropylbenzene)-iron(II) hexafluorophosphate.

Examples of the benzoin tosylate compound include benzoin tosylate and α-methylbenzointosylate.

Examples of the halogen-containing triazine compound include 2-(4-methoxyphenyl)-4,6-bis(trichloromethyl)-1,3,5-triazine and 2-(4-methoxynaphthyl)-4,6-bis. (Trichloromethyl)-1,3,5-triazine, 2-[2-(2-furyl)ethenyl]-4,6-bis(trichloromethyl)-1,3,5-triazine, 2-[2-( 5-Methyl-2-furyl)ethenyl]-4,6-bis(trichloromethyl)-1,3,5-triazine.

Examples of the cyano group-containing oxime sulfonate compound include α-(methylsulfonyloxyimino)-4-methoxybenzyl cyanide, α-(trifluoromethylsulfonyloxyimino)-4-methoxybenzyl cyanide, α-(ethyl Examples thereof include sulfonyloxyimino)-4-methoxybenzyl cyanide and α-(propylsulfonyloxyimino)-4-methylbenzyl cyanide.

Examples of naphthalimide compounds include 6-(n-butylthio)-2-(perfluorobutylsulfonyloxy)-2-aza-2H-phenalene-1,3-dione and 6-(n-butylthio)-2. -(Trifluoromethylsulfonyloxy)-2-aza-2H-phenalene-1,3-dione and 6-(isopropylthio)-2-(trifluoromethylsulfonyloxy)-2-aza-2H-phenalene-1, 3-dione is mentioned.

The photo-acid generator is preferably a naphthalimide compound, more preferably 6-(n-butylthio)-2-(perfluorobutylsulfonyloxy)-2-aza-2H-phenalene-1,3-dione, 6-(n-Butylthio)-2-(trifluoromethylsulfonyloxy)-2-aza-2H-phenalene-1,3-dione and 6-(isopropylthio)-2-(trifluoromethylsulfonyloxy)-2 -Aza-2H-phenalene-1,3-dione.

The content of the photo-acid generator is preferably 0.1 to 30% by weight, more preferably 0.5 to 25% by weight based on the solid content of the photosensitive composition excluding the solvent in order to obtain good patterning performance. %, more preferably 1 to 20% by weight.

The photosensitive composition of the present invention preferably contains a crosslinking agent (particularly, a crosslinking agent that reacts with a reactive group in the presence of an acid). The crosslinking agent may be used alone or in combination of two or more. When there are two or more kinds of reactive groups that react with each other in the copolymer, or when the structural unit (3) has a substituent that reacts with the reactive group, the copolymer has no crosslinking agent. Can be crosslinked with (self-crosslinking). In such a case, the photosensitive composition of the present invention may contain no crosslinking agent.

The cross-linking agent is not particularly limited, and those commonly used in the field of photosensitive compositions can be used in the present invention.

Examples of the cross-linking agent include glycoluril compounds and melamine compounds.
Examples of the glycoluril compound include 1,3,4,6-tetrakis(methoxymethyl)glycoluril, 1,3,4,6-tetrakis(ethoxymethyl)glycoluril, 1,3,4,6-tetrakis( Examples include propoxymethyl)glycoluril and 1,3,4,6-tetrakis(butoxymethyl)glycoluril.
Examples of the melamine compound include hexamethoxymethylmelamine, hexaethoxymethylmelamine, hexapropoxymethylmelamine, and hexabutoxymethylmelamine.

The cross-linking agent is preferably a glycoluril compound, more preferably 1,3,4,6-tetrakis(methoxymethyl)glycoluril, 1,3,4,6-tetrakis(ethoxymethyl)glycoluril, 1, At least one selected from the group consisting of 3,4,6-tetrakis(propoxymethyl)glycoluril and 1,3,4,6-tetrakis(butoxymethyl)glycoluril, and more preferably 1,3,4. , 6-Tetrakis(methoxymethyl)glycoluril.

When a crosslinking agent is used, its content is preferably 0.1 to 40% by weight, based on the solid content of the photosensitive composition excluding the solvent, in order to obtain good patterning characteristics and sufficient temperature response. %, more preferably 0.5 to 30% by weight, still more preferably 1 to 20% by weight.

The photosensitive composition of the present invention contains a solvent. Only one solvent may be used, or two or more solvents may be used in combination. The solvent is not particularly limited, and those commonly used in the field of photosensitive compositions can be used in the present invention.

Examples of the solvent include ethylene glycol monoalkyl ether acetate, propylene glycol monoalkyl ether, propylene glycol dialkyl ether, propylene glycol monoalkyl ether acetate, carboxylic acid ester and its derivative, aromatic hydrocarbon, and ketone.

Examples of the ethylene glycol monoalkyl ether acetate include ethylene glycol monomethyl ether acetate and ethylene glycol monoethyl ether acetate.

Examples of the propylene glycol monoalkyl ether include propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monopropyl ether, and propylene glycol monobutyl ether.
Examples of the propylene glycol dialkyl ether include propylene glycol dimethyl ether, propylene glycol diethyl ether, propylene glycol dipropyl ether, and propylene glycol dibutyl ether.
Examples of the propylene glycol monoalkyl ether acetate include propylene glycol monomethyl ether acetate, propylene glycol monoethyl ether acetate, propylene glycol monopropyl ether acetate, and propylene glycol monobutyl ether acetate.

As the carboxylic acid ester and its derivative, for example, methyl lactate, ethyl lactate, propyl lactate, isopropyl lactate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, isopropyl propionate, butyl propionate, isobutyl propionate, Examples thereof include methyl 3-methoxypropionate, ethyl 3-methoxypropionate, methyl 3-ethoxypropionate, ethyl 3-ethoxypropionate, methyl pyruvate, and ethyl pyruvate.
Examples of aromatic hydrocarbons include toluene and xylene.
Examples of the ketone include 2-heptanone, 3-heptanone, 4-heptanone, and cyclohexanone.

The solvent is preferably propylene glycol monoalkyl ether, more preferably propylene glycol monobutyl ether.

The content of the solvent is preferably 70 to 99.5% by weight, more preferably 50 to 99.5% by weight, based on the photosensitive composition in order to control the film thickness and obtain good patterning characteristics. %, and more preferably 85 to 99% by weight.

The photosensitive composition of the present invention contains components other than the above-mentioned copolymer, photo-acid generator, crosslinking agent and solvent (known surfactants) within a range that does not impair the temperature responsiveness and patterning characteristics in the photolithography method. , Additives, etc.) may be included.

The present invention also provides a cured product obtained from the photosensitive composition. The cured product of the present invention can be produced by a method known per se and the method described below in <Patterned substrate, method for producing the substrate and pattern forming method>.

<Substrate with Pattern, Method for Manufacturing the Substrate, and Method for Forming Pattern>
By patterning the photosensitive composition of the present invention by a photolithography method, a temperature-responsive patterned substrate having a desired response temperature can be manufactured. The present invention also provides such a patterned substrate. The patterned substrate of the present invention may be a substrate having one type of pattern (cured material layer) produced by using only one type of the photosensitive composition of the present invention and having a single response temperature, It may be a substrate having two or more types of patterns having different response temperatures, which are produced by using two or more types of the photosensitive composition of the present invention.

The shape of the pattern is not particularly limited, and when observed from the upper side of the substrate, examples thereof include a square shape, a round shape, a linear shape, and a line and space. When there are two or more patterns having different response temperatures, they may be formed adjacent to each other or may be formed separately. There is no particular limitation on the size of the pattern observed from the upper side and the thickness (height) observed from the pattern cross section. The size of one side of the pattern is, for example, 0.1 μm to 1,000 mm, and the thickness (height) of the pattern is, for example, 5 nm to 1,000 μm. The shapes of these patterns can be controlled by using a plurality of masks having different shapes of transmitted light during the exposure process.

The patterned substrate of the present invention can be manufactured by a method known per se, for example, the method described below. First, the photosensitive composition of the present invention is applied to a substrate by a method such as spin coating or slit coating, and the solvent is removed to form a coating film. When the coating film is irradiated with light such as i-rays (wavelength 365 nm), ultraviolet rays, deep ultraviolet rays, and electron beams through a mask in order to obtain a desired pattern, an acid is generated only in the exposed portion and cross-linking ( (Curing) occurs. After that, it is desirable to perform a post-exposure bake (PEB). The exposed coating film can be developed with a developer or the like to remove the unexposed portion (uncured portion of the coating film) to produce a patterned substrate.

The light for irradiation is preferably ultraviolet light, visible light of 500 nm or less, and more preferably ultraviolet light. The exposure amount is preferably 1~10,000mJ / cm ^2, more preferably 10~5,000mJ / cm ^2, more preferably from 20~3,000mJ / cm ^2.

A substrate having two or more types of patterns having different response temperatures can be manufactured by repeating the above operation for each photosensitive composition having a different composition.

Examples of the material of the substrate include metals, metalloids, metal-containing compounds, metalloid-containing compounds, resins and the like.
Examples of the metal or metalloid include aluminum, nickel titanium, stainless steel (SUS304, SUS316, SUS316L, etc.), silicon and the like.
Examples of the metal-containing compound or the metalloid-containing compound include ceramics, metal oxides or metalloid oxides (glass, silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (nitriding). Silicon, etc.), metal borides, semimetal borides, and the like.

The resin may be any of natural resin, modified natural resin and synthetic resin. Examples of the natural resin include cellulose and the like. Examples of the modified natural resin include cellulose triacetate (CTA) and dextran sulfate-immobilized cellulose. Examples of the synthetic resin include polyacrylonitrile (PAN), polyester-based polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), Polyurethane (PU), ethylene-vinyl alcohol copolymer (EVAL), polyethylene (PE), polyester (PEs), polypropylene (PP), polyvinylidene fluoride (PVDF), polyether sulfone (PES), poly-N-isopropyl. Examples include acrylamide.

<Cell culture support and method for producing the same>
The present invention provides a method for producing a cell culture support, which comprises forming a temperature-responsive cured product layer or pattern on a substrate using a photosensitive composition containing a temperature-responsive polymer, and a method for producing the same. The resulting cell culture support is also provided.

First, a method for producing a cell culture support using one type of photosensitive composition will be described. In the production method of the present invention in this aspect, a coating film is formed on a substrate using a photosensitive composition containing a temperature responsive polymer which is a copolymer containing the structural units (1) to (3), It is characterized by including a step of irradiating the coating film with light to form a cured product layer. The photosensitive composition preferably contains a photoacid generator and a solvent. Further, the photosensitive composition preferably contains a crosslinking agent (particularly, a crosslinking agent which reacts with a reactive group in the presence of an acid). The description of the photosensitive composition (particularly, the type and amount of its components) is the same as the description in the above <Photosensitive composition and cured product obtained therefrom>.

The cured product layer can be formed by a method known per se and the method described in the above <Patterned substrate, method for manufacturing the substrate and pattern forming method>. The thickness (height) of the cured product layer is such that cells are attached to the cured product layer during culturing, and the temperature of the culture system is changed when the cultured cells are collected to separate the cultured cells from the cured product layer. It is preferably 1 to 100 nm, more preferably 2 to 50 nm, and further preferably 3 to 30 nm. The size of the cured product layer observed from the upper side is not particularly limited, and the size of one side of the cured product layer is, for example, 0.1 to 1,000 mm.

The description of the substrate is the same as the description in <Patterned substrate, method for manufacturing the substrate and method for forming pattern>, except for the points described below. The material of the substrate is preferably glass, polystyrene (PS), polyester (PEs), polyethylene terephthalate (PET), polycarbonate, or acrylic resin.

Next, the cell culture support obtained from the method for producing a cell culture support using one type of photosensitive composition will be described. The cell culture support of the present invention in this aspect is formed from a substrate and a photosensitive composition containing a temperature-responsive polymer which is a copolymer containing the structural units (1) to (3) on the substrate. It is characterized by having a cured product layer (that is, a cured product layer containing a cross-linked product of the temperature responsive polymer). The photosensitive composition preferably contains a photoacid generator and a solvent. Further, the photosensitive composition preferably contains a crosslinking agent (particularly, a crosslinking agent which reacts with a reactive group in the presence of an acid). The description of the photosensitive composition (in particular, the type and amount of its components), the cured product layer and the substrate is the same as the above description.

Next, a method for producing a cell culture support using two or more photosensitive compositions will be described. In the production method of this embodiment, a photosensitive composition is used in the method for producing a cell culture support, and the lower limit critical point temperatures of the temperature-responsive polymers contained in two or more photosensitive compositions are different from each other. Is characterized by.

According to the production method of the present invention in this aspect, a coating film is formed on a substrate using a photosensitive composition containing a temperature-responsive polymer (I), the coating film is irradiated with light, and the coating film after exposure is exposed. Is developed to form a pattern, and a photosensitive composition containing a temperature-responsive polymer (II) having a lower critical point temperature different from that of the temperature-responsive polymer (I) is used. The method includes a step (II) of forming a coating film, irradiating the coating film with light, and developing the exposed coating film to form a pattern. The photosensitive composition preferably contains a photoacid generator and a solvent. Further, the photosensitive composition preferably contains a crosslinking agent (particularly, a crosslinking agent which reacts with a reactive group in the presence of an acid).

In the production method of this aspect, one of the temperature-responsive polymers (I) and (II) is a copolymer containing the structural units (1) to (3) (hereinafter abbreviated as “copolymer (123)”. And the other of the temperature-responsive polymers (I) and (II) contains the structural units (1) and (2) and does not include the structural unit (3) (hereinafter referred to as “copolymer”). It may be abbreviated as "polymer (12)"), or the temperature-responsive polymers (I) and (II) are each independently a copolymer (123).

The description of the copolymer (123), the photo-acid generator and the solvent is the same as the description in the above-mentioned <Photosensitive composition and cured product obtained therefrom>. The description of the structural unit (1) and the structural unit (2) in the copolymer (12) is the same as the description in the above <Photosensitive composition and cured product obtained therefrom> except for the points described below. Is.

Each of the structural units (1) and (2) in the copolymer (12) may be of one type or of two or more types. The copolymer (12) may contain a structural unit other than the structural units (1) to (3). The amount of structural units other than the structural units (1) to (3) in all structural units of the copolymer (12) is preferably 20 mol% or less, more preferably 10 mol% or less. The copolymer is more preferably composed of the structural units (1) and (2).
The amount of the structural unit (1) in all the structural units of the copolymer (12) is preferably 10 to 94 mol %, more preferably 20 to 92 mol %, and further preferably 40 to 90 mol %.
The amount (2) of the structural unit in all the structural units of the copolymer is preferably 5 to 40 mol %, more preferably 7 to 30 mol %, and further preferably 10 to 25 mol %.
The copolymer (12) does not contain the structural unit (3).

In the copolymer (12), the structural unit (1) is a structural unit derived from N-isopropylacrylamide, and the structural unit (2) is 2-hydroxyethyl acrylate and N-(2-hydroxy-1-methylethyl). ) A constitutional unit derived from a monomer selected from the group consisting of acrylamide is preferable, the constitutional unit (1) is a constitutional unit derived from N-isopropylacrylamide, and the constitutional unit (2) is 2-hydroxyl. It is more preferable that the structural unit is derived from ethyl acrylate.

The weight average molecular weight (Mw) of the copolymer (12) is preferably 500 to 1,000,000, more preferably 1,000 to 500,000, further preferably 3, in order to obtain good patterning characteristics. 000 to 100,000. This Mw is measured by gel permeation chromatography (GPC) as described above.

When the copolymer (12) is used, its content is preferably 50 to 99% by weight, more preferably 60 to 98% by weight, and further preferably, based on the solid content of the photosensitive composition excluding the solvent. It is 80 to 97% by weight.

The copolymer (12) can be produced by polymerizing a commercially available monomer or a monomer produced by a known method by a known method.

The description of the substrate is the same as the above description in the method for producing a cell culture support using one photosensitive composition.

The pattern can be basically formed by a method known per se and the method described in the above <Substrate with Pattern, Method for Manufacturing Substrate and Pattern Forming Method>.

In order to prevent the pattern formed in the step (I) from being damaged by the solvent contained in the photosensitive composition of the step (II) (that is, the solvent resistance of the pattern formed in the step (I) is For improvement), it is preferable to perform a post-exposure bake (PEB) in the step (I) or further irradiate the pattern formed in the step (I) with light. When step (I) is further performed after step (II), or a step of forming a pattern using a photosensitive composition containing a temperature responsive polymer different from temperature responsive polymers (I) and (II) In the case of performing the step (b), it is preferable to perform the post-exposure bake (PEB) also in the step (II) or further irradiate the formed pattern with light.

The temperature of the post-exposure bake is preferably 40 to 200° C., more preferably 50 to 180° C., further preferably 70 to 160° C., and the time thereof is preferably 0.5 to 1440 minutes, more preferably 1 to 1 minute. It is 600 minutes, more preferably 2 to 480 minutes.

The light with which the formed pattern is irradiated is preferably ultraviolet light, visible light having a wavelength of 500 nm or less, and more preferably ultraviolet light. The irradiation time is preferably 1 to 30 seconds, more preferably 1 to 20 seconds, and further preferably 1 to 10 seconds.

The shape of the pattern may be appropriately set according to the desired shape of the cultured cells. The shape of the pattern can be controlled by using a plurality of masks having different shapes of transmitted light during the exposure process. The thickness (height) of the pattern is preferably set so that the cells adhere to the cured material layer during the culture and the culture cells are separated from the cured material layer by changing the temperature of the culture system when the cultured cells are collected. It is 1 to 100 nm, more preferably 2 to 50 nm, still more preferably 3 to 30 nm. The size of one side of the pattern is, for example, 0.1 to 1,000 mm.

In the production method of this embodiment, only two types of temperature-responsive polymers having different response temperatures (that is, temperature-responsive polymers (I) and (II)) are used to form two types of patterns having different response temperatures. It is also possible to use three or more temperature-responsive polymers having different response temperatures (that is, the temperature-responsive polymers (I) and (II), and one or more other temperature-responsive polymers) to obtain different response temperatures. You may form 3 or more types of patterns.

When using 3 or more types of temperature-responsive polymers, 2 or more types of temperature-responsive polymers are a copolymer (123), and the remainder is from a viewpoint of adjustment of a lower limit critical point temperature (LCST), and maintenance of crosslinkability. It is preferred that one of the temperature-responsive polymers of (1) is a copolymer (12), or that all of the three or more temperature-responsive polymers are independently a copolymer (123).

Next, the cell culture support obtained from the method for producing a cell culture support using two or more photosensitive compositions will be described. The cell culture support of the present invention in this aspect comprises a substrate, and at least one pattern (that is, a temperature-responsive polymer () formed on the substrate from a photosensitive composition containing the temperature-responsive polymer (I). At least one pattern containing the crosslinked product of I)), and at least one photosensitive composition comprising a temperature-responsive polymer (II) having a lower critical point temperature different from that of the temperature-responsive polymer (I). It is characterized by having a pattern (that is, at least one pattern containing a crosslinked product of the temperature responsive polymer (II)). The photosensitive composition preferably contains a photoacid generator and a solvent. Further, the photosensitive composition preferably contains a crosslinking agent (particularly, a crosslinking agent which reacts with a reactive group in the presence of an acid). The description of the photosensitive composition (in particular, the type and amount of its components), the pattern and the substrate is the same as the above description.

The cell culture support of this embodiment has a temperature-responsive polymer formed from two patterns having different response temperatures (that is, two photosensitive compositions containing the temperature-responsive polymer (I) or (II)). It may have only two patterns containing the cross-linked product of (I) or (II), and three or more patterns having different response temperatures (that is, the two patterns and one or more other patterns). Pattern).

<Method for producing cultured cells>
The present invention also provides a method for producing cultured cells using a substrate and a cell culture support having a cured product layer or pattern formed from a photosensitive composition containing a temperature-responsive polymer on the substrate. The description of the cell culture support used in this production method is the same as the description in the above <Cell culture support and its production method> except for the points described below. Further, in the present invention, the method and conditions for culturing cells are not particularly limited, and the cells can be cultured by a method known per se, for example, the method described in Non-Patent Document 1.

First, a method for producing cultured cells using a cell culture support having a cured product layer will be described. In the production method of the present invention in this aspect, a cured product layer formed from a substrate and a photosensitive composition containing a temperature responsive polymer which is a copolymer (123) on the substrate (that is, the temperature response described above). Using a cell culture support having a cured product layer containing a cross-linked product of a reactive polymer, and culturing cells on the cured product layer at a temperature higher than the lower critical point temperature of the temperature responsive polymer. It is characterized by including. The photosensitive composition preferably contains a photoacid generator and a solvent. Further, the photosensitive composition preferably contains a crosslinking agent (particularly, a crosslinking agent which reacts with a reactive group in the presence of an acid). The description of the photosensitive composition (in particular, the type and amount of its components), the pattern and the substrate is the same as the above description.

In the production method of this aspect, after culturing the cells, the temperature of the cured product layer is adjusted to a temperature lower than the lower critical point temperature of the temperature-responsive polymer, thereby making the cured product layer hydrophilic and swelling. Thus, the cultured cells are preferably separated from the cell culture support and collected.

In the production method of this aspect, the cells are cultivated by seeding the cells on a cell culture support and incubating the cells at a temperature suitable for culturing and a temperature higher than the lower critical point temperature of the temperature-responsive polymer. Then, by adjusting the temperature of the cured product layer to a temperature lower than the lower critical point temperature of the temperature responsive polymer, the cells can be detached from the cell culture support and the cells can be recovered.

In the present invention, the cells are not particularly limited, and various cells can be cultured. The cells include, for example, epithelial cells and endothelial cells that form tissues and organs in the body; skeletal muscle cells, smooth muscle cells and cardiomyocytes that exhibit contractility; neurons and glial cells that form the nervous system; fibroblasts. Hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of the living body;

In the present invention, the method of seeding cells is not particularly limited, and a method known per se can be used. For example, a method in which cells are suspended in a medium and seeded on a cell culture support can be mentioned.

In the present invention, the method of collecting the cultured cells detached from the cell culture support is not particularly limited, and a method known per se can be used. For example, cells can be collected by contacting and attaching the cells to a collection membrane. As the recovery membrane, those known per se, for example, collagen gel membrane, cellulose membrane, PVDF membrane, nylon mesh, parchment paper, gelatin membrane and the like can be used.

When the cells are recovered using the recovery membrane, the recovery membrane and the cells can be separated by a method known per se. For example, when a collagen gel film, a cellulose film, a PVDF film, a nylon mesh, or parchment paper is used as the recovery film, by dropping water on the recovery film, the adsorption force between the recovery film and the cells is reduced, Cells can be detached from the membrane. When a gelatin film is used as the recovery film, the gelatin film can be dissolved by heating it to about 33 to 40°C to remove the gelatin film from the cells.

Next, a method for producing cultured cells using a cell culture support having a plurality of patterns having different response temperatures will be described. The description of the production method of this aspect is the same as the description of the production method of cultured cells using the above-described cell culture support having a cured product layer, except for the points described below.

In the production method of the present invention in this aspect, the substrate and at least one pattern (I) (that is, the temperature-responsive polymer (I) formed from the photosensitive composition containing the temperature-responsive polymer (I) on the substrate. Photosensitive material containing at least one pattern (I)) containing a cross-linked product of I), and a temperature-responsive polymer (II) having a lower critical point temperature higher than the lower critical point temperature of the temperature-responsive polymer (I). Using a cell culture support having at least one other pattern (II) (ie, at least one pattern (II) comprising a crosslinked product of a temperature responsive polymer (II)) formed from a functional composition, and A cell (on at least one pattern (I)) at a temperature higher than the lower critical point temperature of the temperature responsive polymer (I) and lower than the lower critical point temperature of the temperature responsive polymer (II). Culturing the cells (I) at a temperature higher than the lower critical point temperature of the temperature-responsive polymer (I) and the temperature-responsive polymer (II) and at least one other pattern (II). And a step of culturing a cell (II) different from the above). The photosensitive composition preferably contains a photoacid generator and a solvent. Further, the photosensitive composition preferably contains a crosslinking agent (particularly, a crosslinking agent which reacts with a reactive group in the presence of an acid). The description of the photosensitive composition (in particular, the type and amount of its components), the pattern and the substrate is the same as the above description.

In the production method of this aspect, after culturing the cells, the temperature of the patterns (I) and (II) is adjusted to a temperature lower than the lower critical point temperature of the temperature-responsive polymers (I) and (II), It is preferable to detach the cells (I) and (II) from the cell culture support by changing the patterns (I) and (II) to be hydrophilic and swelling.

One of the temperature-responsive polymers (I) and (II) is a copolymer (123), and the other of the temperature-responsive polymers (I) and (II) is a copolymer (12), Alternatively, it is preferable that the temperature-responsive polymers (I) and (II) are each independently a copolymer (123).

When the above-mentioned low temperature copolymer is used as the copolymer (123) and the copolymer (12) is used, the low temperature copolymer is used as the temperature responsive polymer (I) having a low lower critical point temperature. It is preferable to use a copolymer and use the copolymer (12) as the temperature-responsive polymer (II) having a high lower critical point temperature.

When the above-mentioned copolymer for high temperature is used as the copolymer (123) and the copolymer (12) is used, the copolymer (12) is used as the temperature-responsive polymer (I) having a low lower critical point temperature. ) Is used, and a copolymer for high temperature is preferably used as the temperature-responsive polymer (II) having a high lower critical point temperature.

In the production method of this aspect, a co-cultured cell sheet formed by contacting cells (I) and (II) with each other by using a cell culture support in which patterns (I) and (II) are in contact with each other. It can be manufactured.

In the production method of this embodiment, a cell culture support having only two patterns having different response temperatures (that is, patterns (I) and (II)) may be used, and three or more patterns having different response temperatures ( That is, a cell culture support having patterns (I) and (II) and one or more other patterns) may be used. Also, as for the cells, only two kinds of cells (that is, cells (I) and (II)) may be cultured, and three or more kinds of cells (that is, cells (I) and (II), and one kind) Other cells described above) may be cultured.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples, and may be appropriately modified within a range compatible with the above and below gist. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

<Production Example 1: Production of monomer>
Under an argon atmosphere, 801 mL of methanol, 100 g of propylamine, 0.02 g of 4-methoxyphenol and 128 g of potassium carbonate were placed in the flask, and the reaction solution was cooled to -20°C. Then, 137 mL of acrylic acid chloride was added dropwise over 30 minutes while adjusting the reaction liquid to -20°C. Then, the reaction solution was stirred at room temperature for 1 hour, and then the reaction was terminated. After distilling off the solvent from the reaction solution, ethyl acetate was added and the salt was filtered. Then, liquid separation extraction was performed with ethyl acetate and water, and the separated organic layer was dried with sodium sulfate. Sodium sulfate was removed by filtration, the filtrate was concentrated, and then distilled under reduced pressure to obtain 121.9 g of N-propylacrylamide.

<Production Example 2: Production of monomer>
Under an argon atmosphere, 2690 mL of methanol, 335.8 g of DL-2-amino-1-propanol, 0.07 g of 4-methoxyphenol, and 340 g of potassium carbonate were placed in the flask, and the reaction solution was cooled to -20°C. Thereafter, 363 mL of acrylic acid chloride was added dropwise over 30 minutes while maintaining the reaction liquid at -20°C. After that, the reaction solution was stirred at -20°C for 3 hours, and then the reaction was terminated. After distilling off the solvent from the reaction solution, ethyl acetate was added and the salt was filtered. Then, liquid separation extraction was performed with ethyl acetate and water, and the separated organic layer was dried with sodium sulfate. After removing sodium sulfate by filtration and concentrating the filtrate, column purification was carried out to obtain 426.6 g of N-(2-hydroxy-1-methylethyl)acrylamide.

<Production Example 3: Production of terpolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 3.6 g of 2-hydroxyethyl acrylate represented by the above formula (2.1), and represented by the above formula (3a.1). A solution prepared by dissolving 7.0 g of N-propylacrylamide and 0.18 g of azobisisobutyronitrile in 26.7 g of propylene glycol monomethyl ether was placed in 44.4 g of propylene glycol monomethyl ether heated under reflux for 2 hours. It dripped over. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a terpolymer. The amount of the structural unit in the terpolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide = 40 mol%, 2-hydroxy Amount of structural unit (2) derived from ethyl acrylate=20 mol %, amount of structural unit (3) derived from N-propyl acrylamide=40 mol %. The weight average molecular weight (Mw) of the obtained terpolymer was 24,900.

The equipment and measurement conditions used for measuring the Mw of the obtained terpolymer are described below. The Mw of a terpolymer or a terpolymer described below was also measured under the same equipment and conditions. Measuring device: HLC-8320GPC [trade name] (manufactured by Tosoh Corporation)
Column: Shodex® KF-803L, KF-802 and KF-801
Column temperature: 40°C
Eluent: DMF
Flow rate: 0.6 ml/min Detector: RI
Standard sample: polystyrene

<Production Example 4: Production of terpolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 1.2 g of 2-hydroxyethyl acrylate represented by the above formula (2.1), and represented by the above formula (3b.1). 0.7 g of N-(2-hydroxy-1-methylethyl)acrylamide and 0.09 g of azobisisobutyronitrile dissolved in 12.9 g of propylene glycol monomethyl ether, and propylene glycol monomethyl heated under reflux. It was dripped into 22.4 g of ether over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a terpolymer. The amount of the constitutional unit in the terpolymer calculated from the amount of the monomer used is as follows: amount of the constitutional unit (1) derived from N-isopropylacrylamide=80 mol %, 2-hydroxy Amount of structural unit (2) derived from ethyl acrylate=13 mol %, amount of structural unit (3) derived from N-(2-hydroxy-1-methylethyl)acrylamide=7 mol %. The Mw of the obtained terpolymer was 35,400.

<Production Example 5: Production of terpolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 1.9 g of 2-hydroxyethyl acrylate represented by the above formula (2.1), and represented by the above formula (3b.2). A solution prepared by dissolving 0.3 g of acrylamide and 0.09 g of azobisisobutyronitrile in 14.0 g of propylene glycol monomethyl ether was added dropwise to 23.3 g of propylene glycol monomethyl ether heated under reflux over 2 hours. did. Then, it was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane and dried in a vacuum oven at 50° C. to obtain a terpolymer. The amount of the structural unit in the terpolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide=75 mol %, 2-hydroxy Amount of structural unit (2) derived from ethyl acrylate=20 mol %, amount of structural unit (3) derived from acrylamide=5 mol %. The Mw of the obtained terpolymer was 18,700.

<Production Example 6: Production of terpolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 1.97 g of 2-hydroxyethyl acrylate represented by the above formula (2.1), and represented by the above formula (3b.3). 0.59 g of N,N-dimethylacrylamide and 0.10 g of azobisisobutyronitrile dissolved in 14.5 g of propylene glycol monomethyl ether were added to 24.1 g of propylene glycol monomethyl ether heated under reflux. It dripped over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a terpolymer. The amount of the structural unit in the terpolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide = 73 mol%, 2-hydroxy Amount of structural unit (2) derived from ethyl acrylate=20 mol %, amount of structural unit (3) derived from N,N-dimethylacrylamide=7 mol %. The Mw of the obtained terpolymer was 19,600.

<Production Example 7: Production of terpolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 1.33 g of N-(2-hydroxy-1-methylethyl)acrylamide represented by the above formula (2.2), and the above formula ( 3.99 g of N-propylacrylamide represented by 3a.1) and 0.12 g of azobisisobutyronitrile dissolved in 18.7 g of propylene glycol monomethyl ether, and propylene glycol monomethyl ether heated under reflux. It was dripped into 31.1 g over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a terpolymer. The amount of the structural unit in the terpolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide=60 mol %, N-( Amount of structural unit (2) derived from 2-hydroxy-1-methylethyl)acrylamide = 10 mol%, amount of structural unit (3) derived from N-propylacrylamide = 30 mol%. The Mw of the obtained terpolymer was 22,700.

<Production Example 8: Production of terpolymer>

21.0 g of N-isopropylacrylamide represented by the above formula (1.1), 2.9 g of 2-hydroxyethyl acrylate represented by the above formula (2.1), and represented by the above formula (3b.1). 4.8 g of N-(2-hydroxy-1-methylethyl)acrylamide and 0.29 g of azobisisobutyronitrile dissolved in 43.4 g of propylene glycol monomethyl ether were heated and refluxed to produce propylene glycol monomethyl. It was dripped into 72.4 g of ether over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a terpolymer. The amount of the structural unit in the terpolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide=75 mol%, 2-hydroxy Amount of structural unit (2) derived from ethyl acrylate=10 mol %, amount of structural unit (3) derived from N-(2-hydroxy-1-methylethyl)acrylamide=15 mol %. The Mw of the obtained terpolymer was 15,000.

<Production Example 9: Production of binary copolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 0.38 g of N-(2-hydroxyethyl)acrylamide represented by the above formula (2.3), and azobisisobutyronitrile A solution prepared by dissolving 0.07 g in 11.2 g of propylene glycol monomethyl ether was added dropwise to 18.6 g of propylene glycol monomethyl ether heated under reflux over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a binary copolymer. The amount of the structural unit in the binary copolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide=95 mol %, N-( Amount of structural unit (2) derived from 2-hydroxyethyl)acrylamide=5 mol %. The Mw of the obtained binary copolymer was 30,400.

<Production Example 10: Production of binary copolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 1.03 g of N-(2-hydroxyethyl)acrylamide represented by the above formula (2.3), and azobisisobutyronitrile A solution prepared by dissolving 0.08 g in 12.2 g of propylene glycol monomethyl ether was added dropwise to 20.3 g of propylene glycol monomethyl ether heated under reflux over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a binary copolymer. The amount of the structural unit in the binary copolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide=80 mol %, N-( Amount of structural unit (2) derived from 2-hydroxyethyl)acrylamide=20 mol %. The Mw of the obtained binary copolymer was 23,300.

<Production Example 11: Production of binary copolymer>

7.0 g of N-isopropylacrylamide represented by the above formula (1.1), 0.89 g of N-(2-hydroxy-1-methylethyl)acrylamide represented by the above formula (2.2), and azobis A solution prepared by dissolving 0.08 g of isobutyronitrile in 11.9 g of propylene glycol monomethyl ether was added dropwise to 19.9 g of propylene glycol monomethyl ether heated under reflux over 2 hours. Then, the mixture was polymerized by heating under reflux for 24 hours. The obtained polymer solution was reprecipitated with hexane, and the obtained precipitate was dried in a vacuum oven at 50° C. to obtain a binary copolymer. The amount of the structural unit in the binary copolymer calculated from the amount of the monomer used is as follows: amount of the structural unit (1) derived from N-isopropylacrylamide=90 mol %, N-( Amount of structural unit (2) derived from 2-hydroxy-1-methylethyl)acrylamide=10 mol %. The Mw of the obtained binary copolymer was 14,000.

[Measurement of Lower Critical Point Temperature (LCST) of Polymer]
The polymers (the terpolymer or the terpolymer) obtained in Production Examples 3 to 7 and 9 were each dissolved in pure water at a concentration of 2% by weight. Obtained by differential thermal measurement using a differential scanning calorimetry (DSC) (“DSC7020” manufactured by SII Nano Technology Co., Ltd.) under a nitrogen atmosphere while lowering the temperature of the aqueous solution at 1° C./min. The temperature at the peak top of the obtained measurement curve was calculated as the lower critical point temperature (LCST) of the polymer. Using the polymers obtained in Production Examples 3 to 11 (terpolymer or binary copolymer), pure water was converted into phosphate buffered saline (Sigma-Aldrich, product number P5493, Phosphate buffered saline, The LCST of the polymer was calculated in the same manner as above except that the concentration was changed to 10x concentrate, BioPerformance Certified, suitable for cell culture). The results of LCST measured using pure water as the solvent are shown in Table 1, and the results of LCST measured using phosphate buffered saline as the solvent are shown in Table 2.

<Example 1: Production of photosensitive composition>
To 2.2 g of the terpolymer obtained in Production Example 3, 0.154 g of a crosslinking agent (1,3,4,6-tetrakis(methoxymethyl)glycoluril), a photo-acid generator (6-(n- (Butylthio)-2-(trifluoromethylsulfonyloxy)-2-aza-2H-phenalene-1,3-dione) (0.33 g) and a solvent (propylene glycol monobutyl ether) (119.3 g) were added to form a photosensitive composition. Obtained.

<Example 2: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the ternary copolymer obtained in Production Example 4 was used instead of the ternary copolymer obtained in Production Example 3.

<Example 3: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the ternary copolymer obtained in Production Example 5 was used instead of the ternary copolymer obtained in Production Example 3.

<Example 4: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the ternary copolymer obtained in Production Example 6 was used instead of the ternary copolymer obtained in Production Example 3.

<Example 5: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the ternary copolymer obtained in Production Example 7 was used instead of the ternary copolymer obtained in Production Example 3.

<Example 6: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the ternary copolymer obtained in Production Example 8 was used instead of the ternary copolymer obtained in Production Example 3.

<Comparative Example 1: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the binary copolymer obtained in Production Example 9 was used instead of the ternary copolymer obtained in Production Example 3.

<Comparative Example 2: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the binary copolymer obtained in Production Example 10 was used instead of the ternary copolymer obtained in Production Example 3.

<Comparative Example 3: Production of photosensitive composition>
A photosensitive composition was obtained in the same manner as in Example 1 except that the binary copolymer obtained in Production Example 11 was used instead of the ternary copolymer obtained in Production Example 3.

[Sensitivity test]
Each of the photosensitive compositions obtained in Examples 1 to 5 and Comparative Example 1 was applied onto a silicon wafer by using a spin coater, prebaked on a hot plate at 80° C. for 1 minute, and applied on the silicon wafer. A film was formed. Then, an i-ray aligner PLA-501 (manufactured by Canon Inc.) was used to expose the coating film while changing the exposure amount. Then, post exposure bake (PEB) was performed in an oven at 80° C. for 20 hours. The post-exposure baked coating film was exposed to isopropyl alcohol for 1 minute, and then rinsed with water at 60° C. for 3 minutes while shaking. Then, the coating film was dried at 60° C. for 10 minutes, and the film thickness of the coating film in the exposed area was measured. The results are shown in FIGS.
Similar results were obtained even after baking after exposure (PEB) at 80° C. for 5 hours.

[Patterning test]
Each of the photosensitive compositions obtained in Examples 1 to 4 and Comparative Example 1 was applied onto a silicon wafer by using a spin coater, prebaked on a hot plate at 80° C. for 1 minute, and applied on the silicon wafer. A film was formed. Then, using an i-ray aligner PLA-501 (manufactured by Canon Inc., exposure amount: 2,000 mJ/cm ² ), exposure was performed through a mask having a line pattern with an interval of 20 μm. Then, post exposure bake (PEB) was performed in an oven at 80° C. for 20 hours. The post-exposure baked coating film was exposed to isopropyl alcohol for 1 minute, and then rinsed with water at 60° C. for 3 minutes while shaking. Then, it was dried at 60° C. for 10 minutes to obtain a patterned substrate. The surface of the patterned substrate is observed using an atomic force microscope (AFM) (“Dimension Icon” manufactured by Bruker AXS KK), and a portion with a pattern (coating film) and no pattern The thickness of the part was measured. The results are shown in FIGS.

From the photosensitive compositions of Examples 1 to 4, substrates having a good pattern (line pattern with a spacing of 20 μm) were obtained (FIGS. 7 to 10), but from the photosensitive composition of Comparative Example 1, the inter-pattern was obtained. However, a substrate having a good pattern could not be obtained (Fig. 11). The reason why the photosensitive composition of Comparative Example 1 could not be patterned well was that the lower critical point temperature (35.4° C., see Table 1) of the binary copolymer of Production Example 9 was changed to that of Production Examples 4 to 6. The crosslinkable structural unit (2) derived from N-(2-hydroxyethyl)acrylamide is added to the same degree as the lower critical point temperature (33.9 to 34.5°C) of the terpolymer of It is possible to reduce it.

<Example 7: Production of cell culture support>
A photosensitive composition containing the photosensitive composition obtained in Example 1 (ie, the terpolymer obtained in Production Example 3 (lower critical point temperature in phosphate buffered saline: 23.3° C.)). Was applied on a 5 cm square glass substrate with a spin coater and heated at 80° C. for 2 minutes (prebaking) to obtain a coating film having a film thickness of 15 nm. Then, the entire area of regions 1 and 2 (the size of each region: about 5 mm×about 3.75 mm) of this coating film is applied to the region 1 by 120 with a liquid crystal projector maskless exposure apparatus λ _peak : 450 to 480 nm. Second, the region 2 was irradiated with ultraviolet rays for 180 seconds (exposure). Then, the exposed coating film is heated at 80° C. for 16 hours (post-exposure bake), immersed in water at 23° C. for 60 seconds and shaken to dissolve and remove the unexposed portions other than the areas 1 and 2. A pattern was formed by (development) and drying at 60° C. for 10 minutes. Then, the obtained pattern was irradiated with ultraviolet rays under the same conditions using the above-mentioned exposure apparatus (re-exposure). Then, the pattern after re-exposure was immersed in 23° C. isopropyl alcohol for 60 seconds, then washed by immersion in 60° C. water for 60 seconds, and dried at 60° C. for 10 minutes to obtain Example 1. A cell culture support 1 having a pattern Ia (corresponding to region 1) and a pattern Ib (corresponding to region 2) formed from the obtained photosensitive composition was obtained.

On the cell culture support 1 obtained as described above, the photosensitive composition obtained in Comparative Example 2 (that is, the binary copolymer obtained in Production Example 10 (in phosphate buffered saline) was used. (Lower critical point temperature: 27.4° C.) was applied by a spin coater and heated at 80° C. for 2 minutes (prebaking) to obtain a coating film having a film thickness of 15 nm. Then, with respect to the entire surfaces of the areas 3 and 4 (the size of each area: about 5 mm×about 3.75 mm) of this coating film, a liquid crystal projector maskless exposure device (λ _peak : 450 to 480 nm) was applied to the area 3 to cover the area 140. Second, the region 4 was irradiated with ultraviolet rays for 200 seconds (exposure). Then, the exposed coating film is heated at 80° C. for 16 hours (post-exposure bake), immersed in 23° C. isopropyl alcohol for 60 seconds, and shaken to dissolve and remove the unexposed portions other than the regions 3 and 4. (Development), washing by immersing in water at 60° C. for 60 seconds, and drying at 60° C. for 10 minutes to obtain the photosensitive composition obtained in Comparative Example 2 in addition to the above patterns Ia and Ib. The cell culture support 2 having the pattern IIa (corresponding to the region 3) and the pattern IIb (corresponding to the region 4) formed from was obtained. FIG. 12 shows a schematic plan view of the patterns Ia, Ib, IIa and IIb in the obtained cell culture support 2. Note that FIG. 12 is a schematic diagram showing the relative positions of the patterns Ia, Ib, IIa, and IIb, and its scale is not accurate.

<Example 8: Production of cultured cells>
Bovine vascular endothelial cells suspended in DMEM medium containing 10% by weight FBS (serum) were added to the cell culture support 2 obtained in Example 7 to give a cell seeding density of 2.5×10 ⁴ cells/cm ^2. The cells were seeded as described above, and cultured for 1 day in a 37° C. incubator (CO ₂ concentration: 5% by volume) to observe the adhered state of the cultured cells. As a result of the observation, it was confirmed that the cells were attached and spread.

Next, the cell culture support 2 to which the cultured cells adhered was held for 60 minutes in an incubator (CO ₂ concentration: 5% by volume) at 20° C., and the detached state of the cells was observed. As a result of the observation, it was confirmed that the cells were desorbed from the surface of the base material and had a spherical shape.

The photosensitive composition of the present invention is useful for producing a temperature-responsive patterned substrate (in particular, a cell culture support) having a desired response temperature.

The present application is based on Japanese Patent Application No. 2014-223734 filed in Japan, the contents of which are incorporated in full herein.

Claims (15)

Formula (1):

(In the formula, R ¹¹ represents a hydrogen atom or a methyl group.
R ¹² and R ¹³ each independently represent a C _1-4 alkyl group.
* Indicates a binding position. )
A structural unit represented by
Formula (2):

(In the formula, R ²¹ represents a hydrogen atom or a methyl group.
R ²² represents a ^{-CO-O-R 23} or ^-CO-NR 24 ^{R 25.}
R ²³ represents a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3 to 10-membered heterocyclic group having a reactive group.
One of R ²⁴ and R ²⁵ is a hydrogen atom, an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3 to 10 Membered heterocyclic group, the remaining one is a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3-10 membered heterocyclic group having a reactive group. Indicates.
* Indicates a binding position. )
And a structural unit represented by the formula (3):

(In the formula, R ³¹ represents a hydrogen atom or a methyl group.
R ³² represents —CO—O—R ³³ or —CO—NR ³⁴ R ³⁵ .
R ³³ is a C _2-10 alkyl group which may have a hydroxy group or —R ³⁶ —R ³⁷ —R ³⁸ (wherein R ³⁶ is a C _1-4 alkylene group, and R ³⁷ is , A C _3-6 cycloalkanediyl group, and R ³⁸ is a C _1-4 alkyl group which may have a hydroxy group .) .
One of R ³⁴ and R ³⁵ represents a C _3-10 alkyl group, and the other one represents a hydrogen atom or a C _1-10 alkyl group .
* Indicates a binding position. )
A copolymer containing a structural unit represented by the following formula (excluding those which are the same as the structural unit represented by the formula (1) and the structural unit represented by the formula (2)):
A photosensitive composition containing a photoacid generator and a solvent. The structural unit represented by the formula (3) includes ethyl (meth)acrylate, propyl (meth)acrylate, isopropyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, sec-butyl (meth)acrylate, tert-butyl (meth)acrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, 1,4-cyclohexanedimethanol mono(meth)acrylate, N-propyl (meth) ) A structural unit derived from a monomer selected from the group consisting of acrylamide, N-butyl(meth)acrylamide, N-(tert-butyl)(meth)acrylamide, and N-methyl-N-isopropyl(meth)acrylamide. A photosensitive composition according to claim 1 . The photosensitive composition according to claim 1 or 2 , wherein the lower critical point temperature of the copolymer is 15°C or higher and lower than 33°C. Formula (1):

(In the formula, R ¹¹ represents a hydrogen atom or a methyl group.
R ¹² and R ¹³ each independently represent a C _1-4 alkyl group.
* Indicates a binding position. )
A structural unit represented by
Formula (2):

(In the formula, R ²¹ represents a hydrogen atom or a methyl group.
R ²² represents a ^{-CO-O-R 23} or ^-CO-NR 24 ^{R 25.}
R ²³ represents a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3 to 10-membered heterocyclic group having a reactive group.
One of R ²⁴ and R ²⁵ is a hydrogen atom, an optionally substituted C _1-10 alkyl group, an optionally substituted C _3-10 cycloalkyl group, or an optionally substituted 3 to 10 Membered heterocyclic group, the remaining one is a C _1-10 alkyl group having a reactive group, a C _3-10 cycloalkyl group having a reactive group, or a 3-10 membered heterocyclic group having a reactive group. Indicates.
* Indicates a binding position. )
A structural unit represented by, and
Formula (3):

(In the formula, R ³¹ represents a hydrogen atom or a methyl group.
R ³² represents a ^-CO-NR 34 ^{R 35.}
R ³⁴ and R ³⁵ each independently represent a C _1-3 alkyl group having a hydrogen atom, a methyl group or an ethyl group, or a hydroxy group .
* Indicates a binding position. )
A copolymer containing a structural unit represented by the following formula (excluding those which are the same as the structural unit represented by the formula (1) and the structural unit represented by the formula (2)):
Photoacid generator, and
solvent
A photosensitive composition containing: The structural unit represented by the formula (3) is acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-ethyl-N-methylacrylamide, N-( The photosensitive composition according to claim 4 , which is a constitutional unit derived from a monomer selected from the group consisting of 2-hydroxyethyl)acrylamide and N-(2-hydroxy-1-methylethyl)acrylamide. The photosensitive composition according to claim 4 or 5 lower critical point temperature of the copolymer is 33-45 ° C.. R ²² Is -CO-OR ²³ And R ²³ Is a C having a reactive group _1-4 It is an alkyl group, The photosensitive composition as described in any one of Claims 1-6. The photosensitive composition according to any one of claims 1 to 7, wherein the reactive group is a hydroxy group. The amount of the structural unit represented by the formula (1) is 10 to 94 mol% and the amount of the structural unit represented by the formula (2) is 5 to 40 mol% in all the structural units of the copolymer. the photosensitive composition according to any one of claims 1-8 amount of structural units represented by the formula (3) is 1 to 80 mol%. Furthermore, photosensitive composition according to any one of claims 1 to 9 containing a crosslinking agent which reacts with the reactive group in the presence of acid. The photosensitive composition according to any one of claims 1 to 10 used to produce the cell culture support. Patterned substrate formed of a photosensitive composition according to any one of claims 1-10. A step of forming a coating film on a substrate using the photosensitive composition according to any one of claims 1 to 10 , irradiating the coating film with light, and developing the coating film after exposure, A method for manufacturing a patterned substrate. A step of forming a coating film on a substrate using the photosensitive composition according to any one of claims 1 to 10 , irradiating the coating film with light, and developing the coating film after exposure, Pattern formation method. Cured product obtained from the photosensitive composition according to any one of claims 1-10.